Method for the devitalisation of natural organs and/or for the preparation of extracellular matrices for tissue engineering

ABSTRACT

74 . Culture medium according to any one of claims  48  to  73 , characterized in that, that autologous growth factors are gained through chemical and/or biochemical destruction of body-own tissues.  
       75 . Culture medium according to any one of claims  48  to  74 , characterized in that, that autologous growth factors are gained through apoptosis of body-own tissues.  
       76 . Culture medium according to claim  74,  characterized by the fact that tissue destruction is done by ultrasound. Summary The invention provides new procedures for the devitalization and preserving of human and animal organs and tissues, preferably however natural hollow organs and all of their components, in particular from blood vessels and cardiac valves. Furthermore the invention provides procedures for the production of matrices for construction of organs and tissues in part or in toto. In addition the invention concerns organs and tissues, in particular natural and artificial hollow organs which can be achieved according to invention-appropriate procedures. Furthermore the invention concerns the clinical use of these organs and tissues an the application in the human and veterinarian medicine, preferably in the cardiac and vascular surgery. Furthermore the invention concerns new culture media.

The invention provides new procedures and processes for thedevitalization and preservation of human and animal organs and tissues,preferably however natural hollow organs and all of their components, inparticular from blood vessels and cardiac valves. Furthermore theinvention provides procedures and processes for the production ofmatrices for the construction of organs and tissues in part or in toto.In addition the invention concerns organs and tissues, in particularnatural and artificial hollow organs which can be achieved according toinvention-appropriate procedures and processes. Furthermore theinvention concerns the clinical use of these organs and tissues and theapplication in the human and veterinarian medicine, preferably incardiac and vascular surgery. The invention-appropriate procedures andprocesses generate organs and tissues which show a higher mechanicalstability and a better suitability for the further procurement by tissueengineering as compared to organs and tissues produced with traditionalprocedures. In particular the immunological compatibility andantithrombogenicity of the processed organs and tissues are asignificantly improved by the invention-appropriate wash out techniqueof unwanted cell decomposition products and cell debris. Such organs andtissues show significantly reduced thrombogenicity and immunogenicity incomparison with the raw materials (organs and tissues) used. The sameapplies to organs and tissues which are only prepared in part accordingto the invention-appropriate procedures, i.e. without theinvention-appropriate wash out step.

Up to now various preservation techniques are used for the preservationand storage of organs and body tissues, designed for reusage in human:

The short-term storage of human cardiac valves for 3 to 4 days in anantibiotic cocktail at 4° C. is carried out, when there is already arecipient for the specific heart valve at the time of harvesting thedonor valve. This technique is known as fresh wet transplantation. Alonger storage of organs using this technique results in destruction ofthe specific organ.

Furthermore the storage of organs and tissues using so calledcross-linking agents, such as glutaraldehyde, formaldehyde, polyetheroxide (polyepoxy compound), hexamethylene or acylazide is known. Anadvantage of this technique is the possibility for the long-term storageafter preparatory treatment with this technique. A disadvantage ishowever the basic non-suitability of such preprocessed tissues for usein body systems which are exposed to a high mechanical stress, as forexample the arterial blood system. Veins and arteries preprocessed insuch a way demonstrate at present a high early graft occlusion rate anda high mechanical failure rate. Experiments trying to detoxifycross-linking agents and recontruct them by methods of tissueengineering have been up to now unsuccessful.

The clinically most important preservation technique of organs andtissues is the cryopreservation.

The cryopreservation and storage of organs and body tissues forpreservation and later use, i.e. within the framework of atransplantation, are known and clinically established. The employedtechniques distinguish in this case only slightly (Brockbank K G M.Basic Principles of Viable Tissue Preservation. In: TransplantationTechniques and Use of Cryopreserved Allograft Cardiac Valves andVascular Tissue. D R Clarke (ed.), Adams Publishing Group Ltd., Boston.S 9-23. American Association of Tissue Banks Standards for TissueBanking (1995), A.A.T.B., McLean, Va., U.S.A. European Association ofTissue Banks General Standards for Tissue Banking (1995), E.A.T.B.,Vienna, Austria).

The cryopreservation is used primarily for the storage of human cardiacvalves, the so-called homografts, and for the storage of human veins orother tissues.

The use of cryopreserved vein allografts for example is an establishedprocedure in bypass surgery (Brockbank K G M et al., Cryopreserved veintransplantation. J. Cardiac Surg. 7:170-176, 1992; Gelbfish J. et al.,Cryopreserved homologous saphenous vein: Early and late patency incoronary artery bypass surgical procedures. Ann. Thorac. Surg. 42:70,1986; Fujitani R M et al., Cryoperserved saphenous vein allogenichomografts: An alternative conduit in lower extremity arterialreconstruction in infected fields. J. Vasc. Surg. 15: 519-526, 1992) andis used in patients, lacking enough body-own vessel material or withvessels of insufficient quality. Such cryopreserved veins are alsofrequently used as bypass grafts in infected body areas. Here the use ofprosthetic material prohibits itself through the high, material-relatedincidence of prosthesis infection.

However, cryopreserved veins show a bad long-term patency rate(Bilfinger T V et al., Cryopreserved Veins in MyocardialRevascularization: Possible Mechanism for Their Increased Failure. Ann.Thorac. Surg. 63: 1063-69, 1997 and comment in Ann. Thorac. Surg. 64:1524-5, 1997. Marshin R S et al., Cryopreserved Saphenous VeinAllografts for Below Knee Lower Extremity Revascularization. Ann. Surg.219: 664-72, 1994). The reason for it is most likely an immunologicaldegeneration of the vein walls (Carpenter J P, Tomaszewski J E,Immunosuppression for Human Saphenous Vein Allograft Bypass Surgery: aProspective Randomized Trial. J. Vasc. Surg. 26: 32-42, 1997. CarpenterJ P, Tomaszewski J E, Human Saphenous Vein Allograft Bypass Grafts:Immune response. J. Vasc. Surg. 27:492-9, 1998). In addition thromboticearly graft occlusions of cryopreserved veins is frequently observed.These two problems have been traced back to damage of the donorendothelium which took place during the cryopreservation process. Thiscan result in the total absence of the endothelium or a limitedfunctioning of the same (Brockbank K G M et al., Cryopreserved veintransplantation. J. Cardiac Surg. 7:170-176, 1992; Brockbank K G M etal., Functional analysis of cyropreserved veins. J. Vasc. Surg.11:94-102, 1990. Laub G W et al., Cryopreserved allograft veins asalternative coronary conduits: early phase results. Ann. Thorac. Surg.54:826-31,1992. Louagie Y A et al., Viability of long term cryopreservedhuman saphenous veins. J. Cardiovasc. Surg. 31: 92-100, 1990).

As a result of this, cryopreservation techniques for allografts andxenografts have been published and already patented that aim toguarantee a high degree of preservation of the donor endothelium. Thisdegree of preservation of the donor endothelium of cryopreserved tissueis given in the literature as being 50-80% (Bambang L S et al., Effectsof cryopreservation on the proliferation and anticoagulant activity ofhuman saphenous vein endothelial cells. J. Thorac Cardiovasc. Surg.110:998-1004).

However a major role has recently been assigned to the endothelium asbeing the culprit for acute and chronic organ rejection. Endotheliumspecific, non HLA antigens which lead to the activation of CD4 T-cellsenables the donor endothelium to supply the recipients immune systemwith foreign antigens in conjunction with other accessory molecules. Therelease of non HLA antigen by damaged endothelial cells leads to achronic immune reaction and possibly to graft vasculopathy and chronicrejection (Rose M L, Role of endothelial cells in allograft rejection.Vasc. Med. 2(2): 105-14, 1997; Reul R M, Fang J C, Denton M D et al, CD40 and CD 40 ligand (CD 154) are coexpressed in microvessels in vivo inhuman cardiac allograft rejection. Transplantation 64(12): 1765-74,1997; Salom R N, Maguire J A, Hancock W W, Endothelial activation andcytokine expression in human acute cardiac allograft rejection.Pathology 30(1): 24-29, 1998). On the other side the selective removalof the donor endothelium results in the absence of acute rejectionreactions in the animal experiment (rat) (Ann. Surg. 206: 757-764,1987), resulting later in spontaneous reendothelialization. In such away preprocessed bypasses showed in the animal experiment improvedpatency rates.

Paying heed to these considerations that immunologically causedrejection reactions origin from all cellulare components of thetransplantats, divers methods were developed for removal of these cells(U.S. Pat. No. 5,613,982). In among others various hydraulytic enzymes(i.e. proteases, lipases, nucleotidases, glycosidases etc.) were used aswell as physical-chemical methods (use of hypotonic media or detergents,steam stage freezing processes, pH extremes etc.) (U.S. Pat. No.5,192,312; U.S. Pat. No. 5,632,778; U.S. Pat. No. 5,613,982, U.S. Pat.No. 5,843,182, WO 95/24873). The main disadvantage of all these methodsis the fact that this treatment may harmfully alter the stability ofother important components for the structural integrity of holloworgans, such as collagens, in particular type I collagen, proteoglycaneor glycoproteine. This is all the more important, since most difficultcomplications which are caused by structural wall weakness of thecryopraeserved veins, such as vein wall tears or aneurysms of the veinwall may lead to complication-prone re-operations, even a long timeafter implantation in humans (Lehalle B et al., Early rupture anddegeneration of cryopreserved arterial allografts. J. Vasc. Surg. 25:751-2, 1997. Couvelard A. et al., Human allograft failure. Hum. Pathol.26: 1313-20, 1995). If decellularized tissue serves forrecellularization procedures as a matrix it becomes necessary, toincubate the tissue supposed to be transplanted with high concentrationsof specific adhesion factors and/or growth factors to facialiate arepopulation of cells in the vessel wall (U.S. Pat. No. 5,632,778 and5,613,982 and/or U.S. Pat. No. 5,192,312, U.S. Pat. No. 5,843,182, WO95/24873). Apart from the fact that it is unknown which influence thehigh non-physiological concentrations of these substances may have onthe functional differentiation of tissue their usage is raising clinicaland legal objections. This is extremely important, since only fullydifferentiated endothelium is effective (Ku D et al., Human coronaryvascular smooth muscle and endothelium-dependent responses after storageat −75° C. Cryobiology 29:199-209, 1992).

It is known at present, that there remains a certain percentage ofviable donor cells after traditional cryopreservation, which has beendiscussed being the culprit for immunological reactions (Yankah A C etal., Antigenicity and fate of cellular components of heart valveallografts. In: Yankah A C, Hetzer R, Yacoub M H, eds. Cardiac valveallografts 1962-1987. Current concepts on the use of aortic andpulmonary allografts for heart valve substitutes. Darmstadt: SteinkopffVerlag 1988). On the other hand other authors consider the viability ofthe transplants for immunologically unimportant and claim a betterendurance of viable transplants (O'Brian M F et al., A comparison ofaortic valve replacement with viable cryopreserved and valves, with noteon chromosomal studies. J. Thorac. Cardiovasc. Surg. 94:812-23, 1987.Angell W W et al., Long term function of viable frozen aortichomografts: a viable homograft valve bank. J. Thorac. Cardiovasc. Surg.93: 815-22, 1987). In general however the viable homograft is preferredtoday. There is a mild rejection reaction in human when ABO compatiblecryopreserved homografts are used for aortic valve replacement. If ABOincompatible homografts are used there is a moderate acute rejectionreaction. In both cases of rejection reactions a T-cell activation wasdetectable for 4-10 days. A clinical relevancy did not exist (Fishingflax T et al., Immunologic reaction and viability of cryopreservedhomografts. Ann. Thorac. Surg. 60: 12-6, 1995).

Except for these disadvantages the findings for the distribution ofantithrombogenic and/or prothrombogenic activities in the wall of holloworgans are up to now hardly considered in the appropriate literature.Since vascular endothelium (covering tissue of the internal- and/orluminal side of all blood vessels and blood vessel valves) ischaracterized by antiaggregatory, anticoagulatory and profibrinolyticactivities (Z. Kardiol. 82: Suppl. 5, 13-21, in 1993; FASEB J. 2:116-123, in 1988), the cellular components of the deeper vessel wall arecharacterized by the expression of tissue factor, which in contact withplasma factors initiate immediately the clotting cascade (ThrombosisRes. 81: 1-41, 1996; J. Clin. Invest. 100:2276-2285, 1997; FASEB J. 8:385-390, 1994; Arterioscler. Thromb. Vasc. Biol. 17: 1-9, 1997). Theprotection from the prothrombotic activities of the wall of holloworgans is of utmost physiological importance not only in blood vesselsand blood vessel valves but also in all others cryopreserved and noncryopreserved natural or artificial hollow organs or vessels.

It it is for example known that the thrombomodulin activity of theremaining donor endothelium is reduced after cryopreservation. Thisleads to a reduction of the anticoagulatory function of the donorendothelium (Bilfinger T V et al., Cryopreserved veins in myocardialrevascularization: possible mechanism for their increased failure. Ann.Thorac. Surg. 63: 1063-9, 1997).

To avoid the disadvantages mentioned above it was early proposed todevelop new or modified organs with improved antithrombotic propertiesby means of tissue engineering (Weinberg C B, Bell E. A blood vesselmodel constructed from collagen and cultured vascular cells. Science.1986;231:397-400.). In doing so, experiences could be used which havebeen gained from endothelial cell seeding on the luminal surfaces ofprosthetic surfaces (Zilla P, R Fasol, Deutsch M, Fischlein T, Minar E,A Hammerle, Krupicka O, Kadletz M. Endothelial cell seeding ofpolytetrafluoroethylene vascular grafts in humans: a preliminary report.J Vasc Surg. 1987;6:535-41.) Zilla et al. were able to prove that astable endothelial cell layer in fact improves the antithrombogenicproperties of these prostheses. A endothelial cell lining of bloodvessels for implantation purposes is today a declared aim ofbiotechnology and tissue engineering.

In vitro studies that we carried out regarding the endothelialization ofcryopreserved allograft veins showed that a precoating of the veins withautologous serum presented an ideal matrix for the cell repopulation ofthe inner surface of the veins. A precoating with patients autologousserum in physiological concentrations promoted not only the adhesion butalso the functional differentiation of the endothelial layer (Lamm P etal. New autologous coronary bypass graft: First clinical experience withan autologous endothelialized cryopreserved allograft. J ThoracCardiovasc Surg 117: 1217-9, 1999). This precoating is superior to aprecoating with with fibronectin with and without a proteoglycan (forexample heparinsulfate) (U.S. Pat. No. 5,192,312; U.S. Pat. No.5,632,778; U.S. Pat. No. 5,613,982, U.S. Pat. No. 5,843,182, WO95/24873, Zilla P. et al., Endothelial cell seeding ofpolytetrafluoroethylene grafts in humans. J. Vasc. Surg. 6: 535-541,1987) because of the shortening of the cell lining procedure and thetotal nonuse of clinically not authorized substances (for examplefibronectin). Since the serum is totally autologous there are noclinical or legal objection.

This invention provides therefore a new, generally applicable method forthe preservation and storage of organs and tissues, in particularhowever of hollow organs. A further task of the invention was to provideorgans and tissues that show a higher mechanical stability and a bettersuitability for the further procurement by the methods of the tissueengineering than the traditional procedures and tissues. In particularthere is a significant improvement in the immunological compatibilityand antithrombogenicity of organs and tissues which are treated by theinvention-appropriate wash out techniques of unwanted cell decompositionproducts and cell debris. Such organs and tissues show in comparisonwith the initially untreated organs and tissues a significantly reducedimmunogenicity and thrombogenicity. The same applies for organs andtissues which have not been treated by the invention-appropriate washout technique.

This task is solved by the patent claims, the following description andthe illustrations.

The present invention describes a procedure or process for thedevitalization and preservation of organs and/or tissues relating tosterile harvesting of organs and tissues until achieving a devitalsteady state in a liquid selected from the group consisting of: sterilewater, a crystalloid liquid, a colloidal liquid, a lipid-containingliquid and a combination of the before mentioned liquids. In the nextstep cell fragments, cellular decomposition products as well as othersoluble substances are washed out under pressure (depending on the organor tissue to be perfused) preferably however using the physiologicnatural organ or tissue specific perfusion pressure, using a liquid or agroup of liquids from the list of liquids mentioned above. The wash outprocess is preferably performed pulsatile, i.e. using variable increaseand decrease pressure curves, depending on the preservation time. Theprocess consists of organ and tissue specific pressure wave forms.Optimization of organ and tissue specific pressure wave forms isdependant on the increase or decrease pressure curves which arenecessary to achieve a devitalization in the respective organ or tissue.The pressure increase velocity, the pressure niveau and the pressuredecrease are adapted to each specific organ or tissue and are optimal,if the wash out of cell debris leads to simultaneous preservation of theextracellular matrix. The preservation of the extracellular matrix andthe successfull wash out of cell debris (cell particles, cell remnants)may be histologically controlled. The condition of these process ischoosen in such a way that it does not prevent at all the formation of aso called collagen-cross-linking. This may be controlled again byhistological examinations.

Furthermore the present invention provides a process for generatingmatrices for the partial or denovo synthesis of organs and/or tissues.This procedure comprises the steps of the devitalization andpreservation of organs and/or tissues according to theinvention-appropriate and of the cell repopularization of organs andtissues, for example by means of reendothelialization. Additionally theinvention provides a cultivation apparatus to be used for theinvention-appropriate procedure.

The invention-appropriate procedure is suitable for the production ofmodified body-own organs and tissues for immediate clinical use of theseorgans and tissues. Arteries and veins for example may be implantedimmediately after the invention-appropriate production process withoutany additional procurement (for example cryopreservation). Theinvention-appropriate produced organs and tissues demonstrate asignificantly higher biomechanical stability than the same organs andtissues after traditional storage and preservation techniques (forexample cryopreservation). In Addition the invention provides processeswhich are suitable to procure invention-appropriate organs and tissuesby the methods of tissue engineering in a clinically safe manner. Inparticular this disclosure provides a process for cell lining of holloworgans with vascular endothelium. These hollow organs are achieved bythe invention-appropriate procedure. In addition theinvention-appropriate procedure may be used to treat organic and/orartificial surfaces, which have been precoated with components of theextracellular matrix (for example collagene, glycosaminoglycane etc.),such as the inner surface of artificial hearts or PTFE- anddacron-protheses, to render a reduced thrombogenicity and immunogenicitywhen compared to the non-preatreated surfaces.

Definitions

“Organ” is defined as a part of the body, which consists of cells andtissues that form a unit with specific tissues. ” The term “Tissue”means, individual kinds of cell groups that have common functions andthat build up the body.

“Invention-Appropriate Organs” are organs of the above definition, thatpassed the invention-appropriate manufacturing-process and which mayonly perform their functions completely or in part after an additionalprocurement by cellrepopulation of the organs, in particular byreendothelialization. The cellrepopulation occurs preferably throughmethods of tissue engineering.

“Invention-Appropriate Tissues” are tissues of the above definition,that passed the invention-appropriate manufacturing-process and whichmay be used clinically with or without a further procurement bycellrepopulation of organs, preferably reendothelialization, inparticular by the methods of tissue engineering.

Tissues, according to the definition are also hollow organs. Holloworgans are for example blood vessels, blood vessel valves, lymphaticvessels, lymphatic vessel valves, cardiac valves, ureters, spermaticducts and bronchial tubes.

“Invention-Appropriate organic- or artificial surfaces” are surfacesthat were precoated with extracellular matrix or matrix components andprocessed with the invention-appropriate procedure.

The term “crystalloid liquid” means every form of buffered or unbufferedsaline solutions. Favoured saline solutions within the framework of theinvention are phosphate buffered saline or clinically authorizedelectrolyte solutions (ringer solution).

The term “colloidal liquid” means protein and/or sugar-containingsolutions. Most preferred solutions are Medium 199 and Brettschneidercardioplegic solution.

The term “lipid-containing liquid” means every form of fat containingsolutions.

The term “dark” means without influence of a natural or artificialsource of light.

The term “under pressure” means perfusion of tissues and organs withinvention-appropriate liquids under application of pressure, dependingon the specific tissue or organ, preferably however under application oforgan and tissue specific pressure values (i.e. the generally knowntypical blood pressure values (=the physiological pressure) for thespecific tissue and organ). In the case of invention-appropriate hollowvessels, a transmural pressure gradient of about 20 to 100 mmHg ispreferred. In the case of complex organs, as for example the liver, thepressure gradient is applicated via the natural blood influx and theorgan environment.

“Steril” means not exposed to germs.

The term “variable flow” means that it is possible to generate differentflow rates in hollow organs by use of the patented cultivationapparatus. This may be used to increase the expression of adhesionmolecules from endothelial cells in the early phase of theinvention-appropriate cell lining of hollow organs with endothelialcells by increasing flow rate and transmural pressure.

The term “Lyophilization” describes a known procedure that is used forthe preservation of labile, altering biological substances. Thesubstances to be dried become frozen mildly in a cooling mixture (forexample carbonic acid snow in methylated alcohol) and are broughtsubsequently under high vacuum (upper boundary of the gap: 0.05-0.1Torr). The ice sublimates and the water steam becomes rapidly removed bya pump supported by suitable hygroscopic means (for exampledeep-freezing-condenser) that in result of the vaporization cold thesubstance to be dried remains in the frozen state.

The term “Critical-Point-Drying” describes a known procedure for thedrying of biological samples. Whereby water gets exchanged by aintermediate medium (for example ethanol). This intermediate medium getsexchanged by carbondioxide. By doing this one takes advantage of thefact that it is impossible above the so called critical point todistinguish between fluid and gaseous stage. It is therfore possible tobypass the direct phase transition fluid-gaseous.

“Antibiotic” means fungal or bacterial metabolism products and theirmodification products with hampering or killing effect against viruses,bacteria and fungals. Gentamicin is one of the preferredinvention-appropriate antibiotics.

The term “Devitalization” means killing of all cells and the reductionof the corresponding organs and tissues to the level of the connectivetissue. This condition is also called “Achieving of Devitalization”.Devitalization can be controlled histologically.

The term “devital steady state” means the state of organs and tissuesafter devitalization. Devitalization is further characterized by thefact that important modulation of the extracellular matrix of organs andtissues, for example the intramolecular cross linking of collagens, isalready advanced and irreversible.

The term “Tissue engineering” describes techniques which allow divers,sometimes organ specific cells to isolate, to cultivate and to propagate(for example reendothelialization of hollow organs such as arteries orveins). Finally new organs and tissues arise from these techniques.

The term “matrix” means scaffold for the reconstruction or themodification of organs and tissues by the methods of tissue engineering.Cells in the tissue culture are propagated onto this “Matrices”.

The term “Repopularisation” means the repopulation with organ ortissue-specific cells, so-called “repopulation cells”.

The term “Apoptosis” (Greek: the dropping of the leaves in the wind)describes a complex process which is also called programmed cell deathand which is used to devitalize tissue and organs. Apoptosis is the mostfrequent form of cell death in the organism. Apoptosis plays anelementary role for the maintaining of the homeostasis of tissues andorgans. The death of individual cells is an essential assumption for thesurvival of the entire organism, because the origin, organization andpreservation of tissues is controlled not only by cell increase anddifferentiation, but it requires controlled removal of damaged cells.Apoptosis is defined by a great number of morphological and biochemicalmodifications. These modifications contain the shrinkage of the cell andthe condensation of the chromatin, that accumulates itself onto theinner side of the nucleus membrane divided specifically intohigh-molecular fragments of 50 and 300 kbp and in many cases in evensmaller fragments of about 200 bp. Specific protein dividing enzymeslike proteases (caspases) are activated to the purposeful removal ofessential proteins for example proteins of the cytosceleton. Thecomposition of the plasma membran changes itself and the cell, inparticular the cell nucleus shrinks, while the cell organells areremaining relatively long functional. Finally the cell dissembles inapoptotic bodies which are removed by phagocytes or neighbouring cells.It is important that the plasma membran remains intact during apoptosisfor the prevention of inflammation reactions.

The term “synthetic material” means every organic and/or anorganicproduct which is suitable for such purposes. In particular the syntheticmaterial is supposed to increase the mechanical stability of theinvention-appropriate organs and tissues.

The invention describes therefore a procedure for the devitalization andpreservation of organs and/or tissues until the invention-appropriatedevital steady state of organs and/or tissues is achieved, including thesterile harvest and storage of the organ or tissue in a liquid, selectedfrom the group consisting of: sterile water, a crystalloid liquid, acolloidal liquid, a lipid-containing liquid or a combination of thementioned liquids and also including the invention appropriate—inparticular pulsatile—wash out of cell debris, cellular decompositionproducts as well as soluble substances under pressure application,preferably by application of physiological pressure, using a liquidselected from the group consisting of: sterile water, a crystalloidliquid, a colloidal liquid, a lipid-containing liquid or a combinationof the above mentioned liquids. Favoured crystalloid liquids inparticular are Bretschneider cardioplegic solution or medium 199(Seromed). Also favoured is a crystalloid liquid which contains anantibiotic. The storage of the organs is done in a preferred techniquein the dark for at least 2 weeks. The storage procedure can also becarried out under light, preferably however under ultraviolettirradiation. This results in the photo oxidation of organs and tissues.The storage in the dark however yields better results.

The harvest of the organ or tissue from dead donors (multi organ donors)is favoured particularly.

In a further, particularly favoured method a multiple rinsing of therespective organs or tissues is done before storage using the sameliquid, which is also used for storage. The storage and wash out of celldetritus, cellular decomposition products and soluble substances can beperformed using the same liquid. It is necessary, that in the case oftissues a pressure gradient across the tissue is made (for example athollow organs a transmuraler pressure gradient, that is pressuregradient across the wall of the hollow organ). In the case of organs apressure gradient is generated between the natural-, organ specificblood, and/or lymphatic vessels and the particular organ to be stored.

Another design of the invention concerns the repeated outwash of celldebris, cellular decomposition products as well as soluble substanceswith organ and tissue-specific calibrated pressure waves (dependent onthe organs and tissues to be treated). Preferably the storage andoutwash of the organs and tissues occurs in a sterile liquid. In aparticularly favoured design of the invention the outwash-procedure forhollow organs is performed in the invention-appropriate culture device(FIG. 1).

In another design of the invention-appropriate procedure the storage ofthe organs and tissues is done for at least 6 months in order to allowthe development of a “devital steady state”. The storage occursparticularly preferred under sterile conditions. If veins are treatedthe particularly preferred invention-appropriate storage time is 6months. Thereafter the outwash of detritus is done with pulsatilepressure.

In another design the storage of the organs and/or tissues is done witha pH-value between 3 and 9, preferred between 6.9 and 7.8, particularlypreferred between 7,0 and 7,5 and with a temperature of 0 to 55° C.,preferred 0 to 37° C., particularly preferred however at 4° C.

In a further design the storage of the organs and tissues is done underreduced oxygen pressure, particularly preferred under anaerobeconditions.

In an additional design the invention-appropriate devitalization and/orstorage is done with gases, which may be in the fluid form (as forexample fluid CO₂), or in gaseous form. The preferred gas is a rare gas.

The invention-appropriate organs and/or tissues can be dried after theinvention-appropriate devitalization and preservation. This drying canbe achieved by lyophilization or by drying at the critical point afterdehydratation.

Organs and tissues that were produced with the invention-appropriateprocedure for the devitalization and preservation show in comparisonwith the native, freshly harvested organs a structurally modified basicframework (extracellular matrix): intermolecular cross-linking and sidechain modifications, which are also called “collagen cross linking”.These organs and tissues are ideal prerequisits—even without specificprecoating—for a partial or total construction of the specific organsthrough means of “tissue engineering”. In addition the organs andtissues, as in the case of the preprocessed blood vessels, may also beimmediately clinically implanted without any further step.

The invention-appropriate procedure allows the devitalization andpreservation of hollow organs, for example blood vessels, blood vesselvalves, lymphatic vessels, lymphatic vessel valves, cardiac valves,ureters, spermatic ducts, bronchial tubes and organs such as bladders,livers, kidneys and hearts.

The invention-appropriate procedure is able to achieve significantimprovements to traditional procedures. The invention-appropriate organsand tissues demonstrate a significantly higher biomechanical stability,as the same organs and tissues after traditional storage andpreservation procedures (for example cryopreservation). Theinvention-appropriately produced blood vessels show a significantlyhigher burst pressure (FIG. 5), in comparison with the same holloworgans after cryopreservation. Of extraordinary importance the fact,that the so preprocessed organs and tissues are not or only littleantigenic or thrombogenic.

Preferred invention-appropriate hollow organs are, if they areimmediately reimplanted, completely deendothelialized and show astaining for keratan sulfate exceeding cell boundaries at otherwiseextensive preservation of the extracellular matrix of the specifichollow organs. This concerns in particular the threedimensionalpreservation of the preserved collagen structures. The slow death of theviable structures during storage and preservation time results even inthe death of such cells, that usually survive cryopreservation (forexample pericytes, “pericyte like cells”). Antigens, which are expressedby these cells, that are still detectable after cryopreservation (in thecase of pericytes and “pericyte like cells” for example the factor whichinitiates the coagulation cascade (“tissue factor”)), are no longerdetecable after the pretreatment with the new procedure.

Another design of the invention intensifies the invention-appropriateprocedure for the devitalization of organs and tissues for example withthe aid of low molecular substances that induce apoptosis directly orindirectly and/or speeds apoptosis up. In a preferred design achemotherapeuticum (for example Methotrexat) is used to induce apoptosisleading to acceleration of the speed of devitalization. The accelerationof devitalization for example by means of low molecular substances canhappen during the invention-appropriate storage of the organ or tissueor in a previous step. Invention-appropriate substances are anysubstances that induces apoptosis directly or decreases or increases theinteraction of signal molecules, which take place in the induction ofapoptosis—in particular the already above-mentioned chemotherapeutica.

Furthermore the invention concerns organs and tissues, in particularhollow organs which are produced with the invention-appropriateprocedure. The hollow organs and in particular the vessels that becomedevitalized and preserved with the invention-appropriate procedure offerideal conditions for their use as organ matrices (so-called scaffolds)in case of tissue engineering and demonstrate in the case of bloodvessels, which have been coated on their luminal surface with patientautologous endothelial cells improved long-term patency rates ascompared to uncoated hollow organs and/or vessels. The organs andtissues for the production of the invention-appropriate matrices for thepartial or denovo construction of organs and/or tissues are ubiquitouslyavailable. The invention-appropriate matrices can be produced withoutany expenditure, they are—in comparison with artificial surfaces—muchless sensitive to infections and less thrombogenic. In addition theproduction can be performed in highest quality by taught, but notespecially educated personnel. In the case of blood vessels the so madeinvention-appropriate blood vessels show the same operative properties(as for example suturability) as untreated native blood vessels. It isalso for the first time possible to implant xenogenic blood vessels inpeople without any further processing after the invention-appropriateproduction. There is the possibility for the industrial transfer of theinvention-appropriate production in the greatest style without anylarger expenditure. The production of a hydrated matrix however isfavoured particularly.

The invention-appropriate procedure provides in the case of complexorgans, as for example liver and kidney, but also in the case of holloworgans, ideal matrices for the creation or the modification of theseorgans and tissues through means of “tissue engineering”.

In the case of complex organs, such as liver and kidney, but also in thecase of hollow organs it is possible to make ideal matrices availablefor the denovo synthesis or modification of such organs and tissues byuse of the invention-appropriate procedure.

Another invention-appropriate procedure concerns about new culture mediathat are characterized by the fact that usual culture media such asbasal media or complex media become supplemented with autologous(patient autologous) growth factors and/or autologous(patient-autologous) adhesion molecules. Suitable basal media are MEMEagle, DMEM, Medium 199, MCDB 131, Ham's medium, Iscore, RPMI (availablefor example through Life Technology, Germany or Seromed, Germany).

The cultivation and propagation of different, partly organspecific cellsis made possible by the invention-appropriate procedure and inparticular by the invention-appropriate culture media. By use of theinvention-appropriate culture media the organspecific differentiation ofthe used cells is maintained and/or initiated. For example well knownproblems regarding the propagation and differentiation of liver cells(hepatocytes) known from the use of traditional cell culture techniquesbecome null and void when invention-appropriate culture media andprocedures are used. That means, it becomes possible to cultivate thecells of the liver (hepatocytes) without any problems wheninvention-appropriate culture media supplemented with autologous growthfactors are used. The same applies for the cells of the kidney. Cellscultivated under these conditions may be used for the modification ordenovo synthesis of organs and tissues, whereby the organs and tissuesinvolved may have been pretreated (for example cryopreservation).Generally these organs and tissues are used as basic frameworks(scaffolds) for their modification through the the above-mentionedcells, through the means of tissue engineering. Ideally by doing thisthreedimensional constructs are produced which are able to take over therespective functions of the organ or tissue to be imitated completely orin part.

Examples of invention-appropriate hollow organs which can bere-implanted immediately after the invention appropriate devitalizationand preservation procedure without any further treatment are human bloodvessels such as arteries and veins. Examples for invention appropriateorgans which may be used as matrices for the denovo synthesis of organsafter the invention appropriate devitalization procedure are amongothers human blood vessels, livers, kidneys, ureters and bladders.

Particularly favoured are invention-appropriate allo- or xenogenicvessels (arteries, veins, lymphatic vessels) with and withoutintraluminal lining with autologous endothelial cells.

Furthermore the invention concerns a procedure for the production ofmatrices for the in part or denovo synthesis of organs, including thesteps of devitalization and preservation of organs and/or tissuesaccording to the invention-appropriate procedure and after achieving theinvention-appropriate devital steady state, the repopularisation ofthese organs and/or tissues, for example by reendothelialization. Theuse of autologous cells (for example endothelial cells) forrepopularization is favoured particularly. Favoured in particular isalso the use of the new invention appropriate culture media,supplemented with autologous growth factors and/or adhesion molecules.

Hollow organs (for example allogenic and xenogenic vessels and ureters)become reendothelialized after devitalization and preservation of theseorgans and tissues. It is for example possible, to use xenogenicmatrices for the construction of a new vessel and its endothelialization(for example bovine mammary arteries that were subjected to theinvention-appropriate procedure).

The invention also concerns about such organs and tissues which havebeen produced by the invention-appropriate procedure, consisting ofdevitalization and preservation as well as reendothelialization.

The invention-appropriate hollow organs that became reendothelializedprior to implantation are characterized preferably by a lining ofpatient autologous endothelial cells on the luminal surface. Aparticularly favoured method of the present invention describes vesselsand their valves, which are lined with recipient autologous endothelialcells on the inner surface (i.e. luminal surface).

Perfusion experiments with invention-appropriate endothelialized donorvessels did not show any differences in endothelial morphology and shearstress stability compared to completely intact, freshly harvested veinsor arteries.

The endothelium of all blood vessels, vascular valves and heart chambersis not only characterized by the above-mentioned antithrombogenicfeatures. It represents an immunologically important barrier—if healthyand intact—against the grossmass of immunologically compentent cells ofthe blood (leucocytes, monocytes, lymphocytes) that pass by withoutdirect contact with the endothelium.

The invention-appropriate procedure allows to produce a completelyconfluent shear stress resistant endothelialcell layer on the luminalsurface of a blood vessel and/or its valves. This layer operates as acomplex antithrombogenic and antiinflammatory catalyst in order toprevent thrombembolism of the hollow organs. Organs, that are coatedwith patient autologous cells, modified or newly constructed, cause notonly no immune reactions at their luminal surface, but reducefurthermore possible immunologic reactions of the deeper vessel wall,preventing any clinically relevant rejection reaction. In contrast forexample to cryopreserved and autologous endothelialized blood vesselsthe new process is able to the first time to completely prevent evenmild rejection reactions. Additionally the new procedure allows for thefirst time a clinically relevant repopulation of complex organs (forexample liver and kidney). Up til now such a repopularization wasimpossible due to the antigenicity of the basal frameword of thesecomplex organs.

Furthermore the present invention concerns the clinical use of complexorgans which were produced according to the invention-appropriateprocedure.

In a particularly favoured invention-appropriate procedure the cellrepopulation, in particular the reendothelialization without anyprecoating of the donor vessels with adhesion factors or serum alone isdone by direct seeding and propagation of the cells, which were producedby use of the invention-appropriate culture medium supplemented withautologous growth factors on the inner graft surface. This is notpossible with any up to now known cell lining technique.

The invention concerns also methods for the production and use of thenew culture media. Invention-appropriate autologous growth factors andadhesion molecules are used for the first time to supplement culturemedia or for the initial processing of organs and tissues which arefurther treated by the methods of tissue-engineering. A favoured usageof culture media which are supplemented with autologous growth factorsand/or adhesion molecules is the precoating of hollow organs prior thecell seeding procedure with autologous growth factors and/or adhesionmolecules. These invention-appropriate culture media is very useful forthe culture of cells from the human circulatory system, in particularfor the culture of vascular endothelial cells. The use of non-autologousgrowth factors or recombinant growth factors is often clinically andlegaly unacceptable for biotechnological applications, i.e. human usage.This is impressively characterisised by the fact that only a fewcommercially available culture media are allowed for human use althoughproven suitability in cell culture. This is due to the fact that humanor animal components are usually added to these media, in particular toserum-free media. Possible liability claims prevent the clinical use ofcomplex growth media in human. The invention-appropriate culture mediaallow the safe culture of human cells.

Examples of basal media, i.e. basal chemically defined culture media fordifferent cell types are: Minimal Essential Medium (MEM) for the cultureof adherent mammal cells (Dulbecco R, G. F. Plaque production by thePolyoma virus. Virology. 1959;8:396-397), Medium 199 for the culture ofmice-fibroblasts or medium RPMI for the culture of tumor cells. Thesemedia differ in the composition of amino acids, vitamins,micronutrients, organic salts and other organic substances which allowthe growth of the cultivated cells.

The term basal media is used synonymously to the term “basal chemicallydefined media”. The term “basal chemically defined media” is used in thetissue culture for culture media of known qualitative and quantitativechemical composition. In contrast to these media, so-called “full media”are supplemented with natural products, such as animal serum.

For the optimal culture of mammal cells basal chemically defined mediabecome supplemented with different sera. Preferably with fetal calfserum (FCS) or Newborn calf serum (NCS) and/or with other growth factorswhich are not exactly defined (for example endothelial cell growthsupplementary: ECGS).

A further aspect of the invention is the supplementation of culturemedia with autologous growth factors, which were produced in differentways, either alone or in combination with autologous serum or incombination with other non autologous growth factors. It doesn't makeany difference whether chemically defined basal media (for example MCDB131) or so-called full media (for example Gibco HE-SFM) arepreferentially used. This results in (1) a faster multiplication ofcells, in particular endothelial cells (see FIG. 2), (2) a significantincrease in the differentiation of the cells and (3) a increase of thelife span of the cells. Another advantage is the absolute clinicalnon-objection of the new culture media, which allow for the first timethe commercialisation of almost all products which are producedaccording to the methods of tissue engineering. This might be a newmilestone for the transfer of organs and tissues which have beenproduced in vitro for the safe use in human.

DETAILED DESCRIPTION OF THE INVENTION-APPROPRIATE CULTURE MEDIA

The new culture media are used for the increase of growth, re-modelingprocesses and reduction of dedifferentiation of vascular cells in cellculture. They are characterised by the fact, that a basal chemicallydefined medium or a full medium is supplemented with autologous(body-own) growth factors and/or adhesion molecules. Theinvention-appropriate culture medium contains beside the basal medium(=basal chemically defined medium) or full medium 5-30%, preferably5-20%, in particular with preference 10-15% autologous serum. Autologousserum means patient-own (obtained by the patient) serum, which containsautologous growth factors and/or autologous adhesion molecules. Inaddition it is preferably not heat inactivated.

In addition recombinant growth factors can be added to theinvention-appropriate culture medium. Examples of suitable recombinantgrowth factors are bFGF, VEGF, EGF, TGF, “Scatter-factor”, PDGF or acombination of these growth factors.

Autologous growth factors and adhesion molecules can be produced fromblood platelets and/or white blood cells. In a favoured mannerautologous growth factors and adhesion molecules are produced fromconcentrated blood platelets. Furthermore the autologous growth factorsand adhesion molecules can be manufactured from clotted whole blood bycentrifugation. Preferably whole blood is stored for at least for 1 hourat 37° C. or for 6 hours at 4° C. (see FIG. 6).

In a further carrying out form glycosaminoglycane can be supplemented tothe invention-appropriate culture medium additionally. Particularlyfavoured glycosaminoglycanes are heparine, heparinsulfate, chondroitine,chondroitinsulfate, dermatine or dermatinsulfate.

In a particularly favoured carrying out form the invention-appropriateculture medium can become supplemented with transferrine,hydrocortisone, insuline, selenium or albumine additionally.

The invention-appropriate culture medium is suitable for the culture ofvascular cells, in particular endothelial cells, perizytes,“pericyte-like-cells” and smooth muscle cells. Furthermore the culturemedium is suitable for the culture of non vascular cells, in particularhepatocytes. In addition the culture medium can be used as a nutritionmedium in tissue engineering applications. In particular it is suitableas a medium for the precoating of vascular prostheses, cardiac valvesand bypass grafts. In a favoured carrying out form theinvention-appropriate culture medium can be used as a preservationsolution in tissue banking.

In a further carrying out form the autologous growth factors can beobtained by mechanical destruction of body-own tissues. Autologousgrowth factors may be obtained in particular by chemical and/orbiochemical destruction of body-own tissues. Autologous growth factorsare particularly favouredly obtained by apoptosis of body-own tissues.Furthermore the destruction of the tissue can be done by use ofultrasound.

In the following favoured procedures for the production of autologousserum are described:

Production of Autologous Serum:

Whole blood is taken from the recipient of the tissue engineered tissuewithout any anticoagulative substances (as for example citrate).Initiation of the clotting process through activation with artificialsurfaces (for example serum tube) or activating substances (for examplethrombin). The serum is gained by centrifugation (400 g for 10 minutes).

Production of Autologous Serum Enriched with Autologous Growth FactorsFrom Blood Cells:

Whole blood is taken from the recipient of the tissue engineered tissuewithout any anticoagulative substances. The initiation of the clottingcascade (see above) results also in the activation of blood cells, inparticular blood platelets and leucocytes. The clotting cascade and thesimultaneous activation of the blood cells can also be initiated throughactivating agents (for example 1IE/ml thrombin). It results in aprogredient liberation of growth factors (for example VEGF, PDGF, FGF).It is known for example that the liberation of VEGF from activated bloodplatelets achieves a maximum after 1 h at 37° C. In order to gain mostof the growth factors, the clotted blood is stored for one 1 h at 37° C.or at least 6 hours at 4° C. Thereafter the growth factors rich serum isgained by centrifugation.

In a further method for the production of autologous serum with a higherconcentration of growth factors from blood cells whole bloodanticoagulated with citrate is taken from the recipient and centrifugedto concentrate the corpuscular components (blood cells). Depending onthe desired degree of enrichment of the blood cells a part of the plasmais removed. After resuspension of the blood cells and recalcification,the clotting cascade is initiated through artificial surfaces or throughphysiological activating agents (for example whole blood or thrombin).Growth-factor-rich serum is gained by the methods described above.

In the following the methods of processing autologous growth factorsfrom blood cells is described

Production of Autologous Growth Factor From Platelets:

Citrate blood is taken from the patient. Platelet-rich plasma is gainedby cautious centrifugation (315 g for 10 minutes). Liberation of theplatelet derived growth factors after recalcification and activationwith essentially whole blood (1 ml on 10 ml dice-rich plasma) whichinitiates the clotting cascade. Concentrated growth factors can beproduced by previous concentrating of the platelets, for example to aconcentration of 2 million platelets/ml and subsequent activation asdescribed above.

Leucocytes were gained by centrifugation of citrated whole blood andisolation of the so-called buffy-coat. Leucocytes were then acivated,for example with FMLP.

In a further processing method concentrated leucocytes and platelets canbe activated together. The isolation of the growth factor rich serum isdone by centrifugation described above.

Isolation of Autologous Growth Factors of Blood Cells and Other TissuesThrough Mechanical Desintegration of the Cells:

In a favoured carrying out form the lysis of the cells occurs throughcomplement activation or apoptosis.

Isolation of Concentrated Autologous Serum or ConcentratedGrowth-Factor-Rich Serum:

Optionally this invention-appropriate serum can be concentrated withstandard concentrators by revocation of water (for example dextranomers,polyacrylic amide). Dextranomers and polyacrylic amide concentrators arecommercially available (Sephadex from Pharmacia, Biogel P from Bio-RadLaboratories). Alternative concentrators as silica gel, zeolite,dextramine, alginate gel, “crosslinked” agarose can be used as well.

The obtained mixture can also be dialyzed against physiologicalsolutions (Hank's salts, Earle's salts, basal media).

The additionally supplementation of basal chemically defined media withheparine, insuline, hydrocortisone, transferrin and selenium next toautologous serum and autologous growth factors is growth promoting. Inparticular heparine is an important co-factor.

The following describes different applications of theinvention-appropriate cultivation apparatus:

The cell lining of invention-appropriate hollow organs can be performedby use of already known bioreactors. The invention-appropriatecultivation apparatus (bioreactor) (FIG. 1) is suited in particular forthis task. The use of the invention-appropriate bioreactor isaccompanied by the following advantages:

There is a constant variable pressure gradient across the vein wall. Inaddition there is transport of medium across the vein wall which is usedfor the nutrition of seeded endothelial cells and—if wanted—of othercells which have been seeded in the vein wall. In addition antigens ofthe vessel wall will be washed out into the outer medium. In additionthe invention-appropriate bioreactor allows a continuous perfusion otthe endothelialized hollow vessels if it seems necessary to supportcertain stages of differentiation of the cells. It results in a clearlyincreased synthesis of extracellular matrix proteins which supports theshear stress stability of the seeded endothelial cell layer of theendothelialized hollow organs. This device is simple, easy to handle,cheap and a safe tool for any endothelialization procedure of holloworgans of every kind.

Furthermore the invention-appropriate bioreactor is suitable also forfollowing processes:

-   -   for repopulation of prosthetic and organic material, in        particular for the reendothelialization with and without        perfusion of the hollow organ.    -   in modification (FIG. 3), for the outwash of unwanted soluble        and non-soluble substances from prosthetic and organic material,        in particular from hollow organs. The outwash is done by        application of a transmural pressure gradient.

It is possible to apply the invention-appropriate procedure for theproduction of matrices for the partial or denovo synthesis of organs andtissues to all natural and artificial hollow organs and theircomponents, for example to natural blood vessels, blood vessel valves,lymphatic vessels, lymphatic vessel lids, ureters and bladder, spermaticducts, bronchial tubes, the heart and in particular cardiac valves.

During the production of so-called biological cardiac valves ofxenogenic materials (for example from bovine pericardium) the rawmaterial used is often fixed with so-called crosslinking agents (forexample glutaraldehyde). By doing so the possible storage duration ofthe raw materials extends. In the next step the raw materials are placedonto so-called “stents” in order to achieve a biological form andbiomechanical stability. These “stents” serve also as supports for theanchoring of the surgical sutures during the implantation. It is knownthat there are chronic immunological processes after implantation ofsuch cardiac valves which finally results in degeneration of theaffected cardiac valve. The stability of such cardiac valves afterimplantation in people is known to be no longer than 15 years. Afterthat a replacement of the cardiac valve must be carried out in a redooperation with a significantly higher operation risk for the affectedpatient. Structure antigenes of the initial tissue employed to thecardiac valve production are causal for these immunologic processes. Ifthe invention-appropriate raw materials are used such antigenicstructures become completely eliminated resulting in increased lifespans of biological cardiac valves. Invention-appropriate cardiac valvescan be implanted without any further treatment. It is also possible tofurther procure these valves with any other preservation solution ortechnique which is already in use for the production of biologic heartvalves.

We demonstrated, that there is no difference of the mechanical stabilityof coated or uncoated invention-appropriate hollow organs after 12 monthof storage or freshly harvested donor veins (Burst pressure test andhistological examinations of the extracellular matrix). Theinvention-appropriate vessels showed however a higher mechanicalstability than cryopreserved vessels (FIG. 5).

The preferred usage of the invention-appropriate procedure is theproduction of matrices for partial or total synthesis of organs andtissues from donor vessels (veins or arteries), as well as xenografts. Aspecial advantage of the procedure is the possibility of antiviraltreatment of the vessels prior to coating. This is possible since thevessel wall of the invention-appropriate vessels shows a by far highermechanical stability than for example the wall of a cryopreservedvessel.

In a favoured invention-appropriate technique the re-popularization ofthese organs with cells occurs after invention-appropriate outwash in anorgan-specific threedimensional rotation equipment. Such rotationequipment is commercially available (Rotary Cell Culture System™,Synthecon Inc, USA).

It is also possible to use vessels in an invention-appropriate manner,which are reendothelialized with patient autologous endothelial cells,whereby the endothelial cells are of different origin (for exampleperipheral blood, bone marrow, fat tissue, gene-technically modified orproduced endothelium, xenogenic and gene-technically modified xenogenicendothelium).

In addition patient-autologous epithelium can be producedgene-technologically in such a way that epithelium is produced whichimitates the surface- and immunological properties ofpatienten-autologous epithelium.

In another invention-appropriate method (see detailed description inexamples 15-17) precoating of artificial surfaces is performed withcells, which are able to produce extracellular matrix. In a second stepsurfaces, which have been treated in this manner, are further procuredby the invention appropriate methods.

Further procurement of surfaces which have been produced in this mannermay be done by the methods or processes of tissue engineering or themethods or processes of anorganic or organic chemistry (for example bychemical insertion of antithrombotic features. Furthermore it ispossible to treat the surfaces with supporting substances, such asadhesion molecules.

In another method the invention-appropriate hollow organ is in additionenclosed on the exterior surface from a coat of a synthetic material.This synthetic coat can consist of resorbable material, for example ofsynthetic polyglyconacid. Hollow organs which are surrounded from a coatof synthetic material show the advantage that they are stabilized forseveral months.

In Comparison with up to now published results regarding to theantithrombogenicity of connective tissue the invention-appropriateorgans and tissues show a significantly reduced thrombogenicity. FIG. 4a and b show an invention-appropriate vein that was explanted 16 hoursafter implantation into a patient. The vein shows a completely smoothsurface without any adhesion of fibrin, Platelets, and leukocytes. Thepatient autologous arteries and veins which had been employed duringthis operation as well were all covered on the inner surface withadhesion deposits of platelets and leucocytes and (FIG. 4C) werecompletely thrombosed.

The invention-appropriately produced organs can be employed in theentire field of medicine and veterinarian medicine, in surgery, inparticular in heart and vascular surgery. Uncoated or coatedinvention-appropriate vessels find special use as aortocoronary bypassgrafts in patients with coronary heart disease and as vessel transplantsfor vessel reconstructions of any kind. This concerns for example theperipheral arterial occlusion disease, aneurysmatic changes in thevessel wall that require replacement of the respective vessels as wellas all cardiac and vascular redo operations. In particular these vesselsare the ideal conduit for use in infected body areas. Furtherindications for use of such vessels represent a great number ofcongenital deformations (for example any form of shunt operations). Inaddition such vessels may be used for basic research as for example ofarteriosclerosis research or permeability studies of pharmaceutics. Thepossibility to be able to implant uncoated vessels at any time withoutany further precurement is of special interest. This also allows the inhospital storage of these vessels in the same manner as the usualstorage of artificial protheses. The invention-appropriatemanufacturing-process makes therefore for the first time in the historyof medicine an alternative bypass for use in cardiac surgery available,that can be used at any time.

The figures are used for the explanation of the invention.

FIG. 1 shows a bioreactor for the invention-appropriate procedure: itconsists exclusively of biologically inert, sterilizeable parts. By useof this device a variable pressure gradient across the vein wall can bebuilt up. Furthermore the vessel can be perfused under pressure by theaid of a pump.

The bioreactor consists of a culture vessel filled with medium (1) inwhich the hollow organ is (for example a vein (2)) transferred. Thelumen of the two vein ends is connected with the two outflows of theculture vessel by means of two adapters (3). One adapter is connectedwith a computer controlled peristaltic pump (7). The other adapter endsat an stock or dump vessel (5) with a riser tube (4). If the vessel (5)represents a dump vessel, the hose (6) must be connected to a stockvessel. The pressure gradient Δp (dependent on the riser tube (4) andpressure transducer (8)) is tuned after desired pressure gradient aboutthe vessel wall. If the pressure educated by the stalk (4) issufficient, the apparatus can also be used without pressure transducers(8). Through the peristaltic pump (7) it is possible to perform a changeof medium in the inside lumen of the vein or a continuous/discontinuousperfusion of the vessel (2). Pressure pipe (9), entrance ports (10),sterile filters (11).

FIG. 2 shows the growth behavior of cultivated human saphenousmacrovascular endothelial cells under different culture conditions(daily 50% media change).

-   -   a) Cultivation of the cells in medium MCDB 131 with 20% pool        serum.    -   b) Cultivation of the cells in medium MCDB 131 with 20%        autologous serum.    -   c) Cultivation of the cells in medium MCDB131 with 20% pool        serum+10 ng/ml rbFGF.    -   d) Cultivation of the cells in medium MCDB131 with 20% of        invention-appropriate growth factor rich autologous serum (of        platelet-rich plasma: 2 million platelets/ml).

It is clear that the invention-appropriate medium (d) offers the bestculture conditions for human endothelial cells by far.

FIG. 3 shows a modification of the bioreactor shown in FIG. 1 for theinvention-appropriate pressure-dependent flushing system of a holloworgan. In this case no media recirculation occurs (compare FIG. 1). Thestock vessel I (11) includes the liquid which is applied under pressure(dependent on the riser tube (4) and pressure transducer (9)) to theinner lumen of the hollow organ (2) with aid of a pump (7) and collectedin the dump vessel I (5). The stock vessel II (12) contains the liquidthat is used, with the aid of the pump (8), to circulate the outermedium of the hollow organ (2). The liquid, including the via the wallof the hollow organ filtered liquid is then collected in the dump vesselII (6). Pressure pipe (10), entrance ports (13), sterile filter (14).

FIG. 4 shows a invention-appropriate vein after implantation incomparison with an autologous native vein. FIG. 4 a and 4 b shows theinvention-appropriate vein that was taken by autopsy 16 hours afterimplantation into a patient. In histological evaluation of the innersurface of the vein there was a completely smooth surface without anyadhesion of fibrin, platelets and leucocytes. In contrast FIG. 4 c showsthe inner surface of the autologous native vein which is also used asbypass material during this operation. It was completely occluded bythrombosis and showed in histological evaluation of the inner surfaceadhesions of fibrin, platelets and leucocytes.

FIG. 5 shows the burst-pressures of a) freshly harvested veins, b)cryopreserved veins immediately after thawing and c)invention-appropriate veins 12 months after invention-appropriatestorage.

The following examples explain the invention and are not to beunderstood as limiting.

EXAMPLE 1 Devitalization and Preservation of Blood Vessels

Donor veins are taken according to traditional technique by the organdonor sterilely. These vessels are examined in the operating room fortheir integrity. Possible side branches of the veins become ligated withsuture material (for example Ethibond 4/0) in usual technique. Thevessels are rinsed multiple with cristalloid solution (for exampleBretschneider cardioplegic solution or medium 199 (Seromed)) andtransferred into a small tube from approx. 1 cm caliber (a sterile epoxytube or a special developed glass tube can be used). The vessel isfilled with medium 199 and stored in the dark at 4° C. Optionally theveins can also be filled with medium 199, shut at both ends for examplewith vessel tie-clips and stored then in medium 199. This has theadvantage that the vessel does not collapse if it is withdrawn from itscontainer. The storage should last at least 6 months. The vessel after astorage time of more than 24 months is still suitable for itsinvention-appropriate use without any limitation. After verification ofsterility by microbiological tests and invention-appropriate flushing ofthe vessel after withdrawal from the storage container the vessel can beimplanted immediately. The inner surface of the hollow organ can besmoothed before the implantation mechanically. To do so a standardballoon catheter (Fogarty-catheter) can be pulled through the holloworgan. This procedure is recommended since storage-conditionalunevenness of the surface can not be excluded.

EXAMPLE 2

Invention-appropriate preparation of donor veins in accordance withexample 1 with a storage time of 6 months. A devitale “steady state” isreached by this procedure.

EXAMPLE 3

Invention-appropriate procedure in accordance with example 2 with apH-value of 7,0 and a temperature of 18-22° C.

EXAMPLE 4 Patient-Autologous Endothelialization of Invention-AppropriateModified Veins

Approx. 500 ml of whole blood is taken from the patients prior to theoperation without any anticoagulative substances. Whole blood is thenstored at 4° C. for 24 hours and then centrifuged to gain serum.Afterwards the serum is deep-frozen up to further use.

In a first operation a 5 cm long autologous vein remnant is taken fromthe recipient in local anesthesia. The cell isolation and furtherpropagation of endothelial cells is done according to usual cell culturetechniques (Jaffe E A, Nachman R L, Becker C G, et al. Culture of humanendothelial cells derived from umbilical veins. Identification bymorphologic and immunologic criteria. J Clin Invest 52:2745-56, 1973).Medium 199 (Seromed) supplemented with 20% autologous serum and 2 ng/mlrecombinant bFGF (basic fibroblast growth factor) can be used as culturemedium for example. After a sufficient cell number for theendothelialization procedure is reached, a invention-appropriate donorvein is taken from its storage container. The vein becomes directlywithout any pretreatment, filled with the endothelial cell suspension(see later in this chapter) or is pretreated by incubation of the innersurface of the vein with patient-autologous serum in a incubator for 12to 24 hours at 37° C. For this purpose both ends of the vein areconnected with an adapter which is closed by use of a reusable stuffing.After removal of the serum by opening the stuffing the precoated vein isfilled with a defined cell number (80.000-120.000 cells/cm² inner graftsurface) of patient-autologous endothelial cells and locked throughreintroduction of the stuffing. For homogenous seeding of theendothelial cells to the inner surface the vein becomes rotated forseveral hours in a rotating device (Kadletz M, Moser R, Preiss P, et al.In vitro lining of fibronectin coated PTFE grafts with cryopreservedsaphenous vein endothelial cells. Thorac Cardiovasc Surg, 35 Spec No 2:143-147, 11/1987) in a incubator at 37° C. After homogenous seeding thevein is taken from the rotating device and transferred into the specificbioreactor (see FIGS. 1 and 3).

EXAMPLE 5 Patient-Autologous Endothelialization of Invention-AppropriateModified Veins Under Use of Patient Autologous Growth Factors andAdhesion Molecules:

The endothelialization of invention-appropriate preprocessed allograftswas carried out according the procedure described in example 2. Incontrast to example 2 invention-appropriate autologous growth andadhesion-factor-rich serum was used in the pretreatment of theallografts. For the cultivation of the cells culture medium substitutedwith invention-appropriate autologous growth-factor-rich serum (MCDB131+20% invention-appropriate serum) is used.

Production of the autologous growth and adhesion-factor-rich serum:

Approx. 500 ml of whole blood supplemented with anticoagulativesubstances favouring citrate is taken from the patient prior to theoperation. Isolation of platelet-rich plasma (plasma with concentratedplatelets) through cautious centrifugation (315 g for 10 minutes).Liberation of the autologous growth factors by degranulation of theplatelets after recalcification and activation with whole blood (1 mlwhole blood for 10 ml platelet-rich plasma). Concentrated growth factorscan be produced by previous concentrating of the platelets, for exampleto a concentration of 2 million platelets/ml and subsequent activationas described above.

Optionally the invention-appropriate serum can be concentrated withstandard concentrators by revocation of water (for example dextranomers,polyacrylic amide). Dextranomers and polyacrylic amide concentrators arecommercially available (Sephadex from Pharmacia, Biogel P from Bio-RadLaboratories). Alternative other concentrators as silica gel, zeolite,dextramine, alginate gel, “crosslinked” agarose can be used.

The obtained mixture can also be dialyzed against physiologicalsolutions (Hank's salts, Earle's salts, basal media).

EXAMPLE 6 Patient-Autologous Endothelialization of Invention-AppropriatePreprocessed Xenografts:

The endothelialization of invention-appropriate preprocessed xenograftsis carried out according to the procedure described in example 2 or 3.

EXAMPLE 7 Patient-Autologous Endothelialization of Another Vessel, forExample an Artery

The endothelialization process of an artery is carried out in the sameway as shown by the endothelialization procedure of a vein described inexample 2 or 3.

EXAMPLE 8 Epithelialization of Another Hollow Organ, for Example anUreter.

The epithelialisation of an ureter is described correspondingly to theendothelialization procedure shown in the example 2 with the difference,that instead of endothelium urothel is used.

EXAMPLE 9 Endothelialization Procedure with Isolated Endothelial CellsFrom Other Origin

Endothelialization procedure as described in example 2. The isolation ofthe corresponding endothelial cells is done from peripheral blood, bonemarrow and abdominal fat according to well known methods. The isolationof that kind of endothelial cells has a clear benefit for patients sincethese procedures are also available for such patients which do not haveany sufficient vascular substrate for the isolation of autologousendothelial cells. In addition these procedures are less invasive forthe patient.

The following examples refer to the use of the invention-appropriatemedia (media supplemented with autologous growth factors and adhesionmolecules) in terms of cell culture technique for tissue engineering.

EXAMPLE 10 Isolation and Cultivation of Human Macrovascular EndothelialCells (From Veins and Arteries):

The isolation is done as described above according to the method Jaffeet al. The cultivation of the cells is done preferably in medium MCDB131 with 20% autologous growth factor rich (from platelets) serum (2×10⁶platelets/ml) substituted in addition with Heparin (50 μg/ml).

EXAMPLE 11 Isolation and Cultivation of Human Smooth Muscle Cells FromMedia Pieces of the Aorta

Primary culture of aortic smooth muscle cells can be obtained by atleast two methods:

-   -   1. outgrowth of smooth muscle cells from explanted pieces of the        aortic media    -   2. enzymatic dispersion of the aortic media. Method 2 is        favoured due to higher cell gains.

The media of pieces of the human aorta is separated from the intima andadventitia surgically. The separated media contains no fragments ofintima or adventitia. The media is mechanically dissected in 5 mm piecesand subsequent incubated with a proteolytic solution (0.05% elastasetype III, 0.225% collagenase, 1% human albumine in phosphate bufferedsaline). One gram tissue is digested with at least 10 ml of enzymesolution. The tissue is incubated at 37° C. until practically completedispersion, which usually required 3-5 h. After complete digestion thecell suspension is filtered through a nylon mesh (50 μm), centrifuged(190 g, 10 min) and resuspended in autologous culture medium (M199+20%autologous growth-factor-rich serum). Cells were seeded at a density of10⁴ cells per 1 cm² in tissue culture plastic and cultured at 37° C. ina humidified atmosphere of 5% CO₂ and 95% of air.

EXAMPLE 12 Isolation and Cultivation of Humane Keratinocytes

For the isolation of humane keratinocytes, skin remnants from operationscan be used (for example circumcision). For the transportation of theskin remnants to the laboratory a basal medium (for example DMEM)substituted with antibiotics (for example Gentamicin 50 ng/ml) for thereduction of the natural skin flora and prophylaxis of a secondaryinfection is used.

At first hairs and necrotic tissue is removed from the skin remnant.Afterwards that fat tissue and vessels of the subcutis are separatedcautiously. For the dispersion of the keratinocytes the preparated skinis placed in a trypsin/EDTA-solution (0.25%/0.2%) for 18 hours at 4° C.After 18 hours of incubation the enzyme action is visible by the factthat the skin becomes a jelly-like state. In order to wash out theremaining trypsin/EDTA solution, the skin is rinsed with phosphatebuffered saline. After that the dissociated tissue is removed from theskin and suspended in culture medium. This cell suspension is filteredthrough a sterile mull compress (or a 50 μm nylon mesh) to remove tissuefragments and debris. The cell suspension is centrifuged (190 g, 10 min)and resuspended in autologous culture medium. Then the cells are seededin tissue culture plastic. In a humidified atmosphere of 5% CO₂ and 95%of air and 37° C. temperature the primary culture is kept for 24 hoursin the incubator. After 24 hours of incubation in which the cells canadhere to the culture dish the medium is changed. After that the culturemedium is changed every 3 days.

As a culture medium the culture medium MCDB 153 (Boyce S T, Ham R G.Calcium-regulated differentiation of normal human epidermalkeratinocytes in chemically defined clonal culture and serum-free serialculture. J. Invest Dermatol. 1983;81:33s-40s) substituted with insuline(5 mg/l), hydrocortisone (1.4 μM, 0.5 mg/l), ethanolamine (0.1 mM),phosphoethanolamin (0.1 mM) with 10% autologous serum and 10% autologousgrowth factors from platelets (2×10⁶ platelets/ml) can be used.

EXAMPLE 13 Isolation and Cultivation Human Dermal Fibroblasts

After isolation of the keratinocytes the remaining skin is given into aculture bottle with autologous growth-medium (M199 with 10% autologousgrowth-factor-rich serum). After few days incubation in the incubator(5% CO₂, 37° C.) fibroblasts growths out from the skin. After sufficientoutgrowth of fibroblasts from the skin, the remaining skin is removed.Change of culture medium is performed every 3 days.

EXAMPLE 14 Cultivation of Human Hepatocytes

Isolation of human hepatocytes is performed according to the method ofBerry et al. (Berry M N et al., High-yield preparation of isolatedhepatocytes from rat liver. In Laboratory Techniques in Biochemistry andMolecular Biology, vo. 21 (ed. R H Burdon and P H van Knippenberg), pp.15-58. Elsevier: Amsterdam, New York, Oxford, 1991).

The cells were seeded in a density of 1.6×10⁵ cells/cm² in culturebottles. As a culture medium medium William's E medium (Gibco, GrandIsland, N.Y., USA) substituted with 15% autologous growth factor- andadhesion molecule-rich serum (growth factors from 2×10⁶/ml platelets and7 ×10⁵/ml leucocytes), 25 mM HEPES, 5 μg/ml insuline, 0.5 μg/mlhydrocortisone, 5 μg/ml transferrin, 100 U/ml penicillin, 100 μg/mlstreptomycin is used. 50% of the culture medium is changed every 24hours.

EXAMPLE 15 Coating of an Artificial Surface (Here PTFE Prosthesis, Ø 4mm) With Fibroblasts and Subsequent Invention-AppropriateAftertreatment.

Isolation and cultivation of fibroblasts is described in example 13. Thesterile PTFE-prosthesis to be coated is inserted into autologous serum.The inner lumen of the prosthesis must be wetted completely with theserum. The prosthesis is stored then for approx. 12 hours at 37° C. Thenthe prosthesis is taken and filled with a cell suspension of fibroblasts(100000 cells/cm² inner prosthesis surface). The prosthesis is nowrotated for 6-10 hours in a rotating device (see also example 4)guaranteeing a homogenous seeding of the cells. Then the coatedprosthesis is transferred into a bioreactor and cultivated for 4 weeksuntil a layer of extracellular matrix of a thickness of at least 10 μm,fitting to the inside lumen firmly, is formed. Then the coatedprosthesis is treated invention-appropriate to reach the devitale steadystate. After invention-appropriate treatment of the prosthesis it can beimplanted immediately or further procured within the framework of tissueengineering.

EXAMPLE 16 Seeding and Coating of an Artificial Surface (HerePolyurethane Prosthesis, Ø 4 mm) With Cells of Subintimal Origin andSubsequent Invention-Appropriate Aftertreatment.

The isolation of subintimal cells is performed from vessels after theproteolytic isolation of the endothelial cells (see example 10). Thesubintimal cells are detached from the vessel by a further subsequent15-minute proteolytic desintegration of the remaining cells (=subintimalcells) on the inner surface of the vessels by use of the sameproteolytic solution (collagenase). After proteolytic dispersion of thesubintimal cells the cells are collected by rinsing the vessel. Thecells are cultivated as described in example 13. The further procedureis done according to example 15.

EXAMPLE 17 Endothelialization of a Invention-Appropriate PreprocessedCoated Artificial Surface (Here PTFE Prosthesis, Ø 4 mm).

PTFE prosthesis were prepared according to example 15. After the devitalsteady-state is reached the treated PTFE prosthesis is endothelializedwith the method shown in example 4.

1. Procedure or process for devitalization and preservation of organsand/or tissues, comprising the following steps: a. sterile harvest andstorage of the organ or the tissue in a liquid selected from the groupconsisting of: sterile water, a crystalloid liquid, a colloidal liquid,a lipid-containing liquid or a combination of the mentioned liquids,until the devitalization is achieved, b. the outwash of cell-fragments,cellular decomposition products (debris) as well as soluble substancesunder pressure with a liquid, selected from the group consisting of:sterile water, a crystalloid liquid, a colloidal liquid, alipid-containing liquid and a combination of the mentioned liquids. 2.The procedure or process of claim 1 wherein the sterile harvest of theorgan and/or tissue is performed from dead people (multi organ donors).3. The procedure or process of claim 1 wherein the storage occurs for atleast 2 weeks, preferably for 6 weeks, in particular preferred howeverfor 6 months in the dark.
 4. The procedure or process of claim 1 whereinthe storage occurs under sterile conditions.
 5. The procedure or processof claim 1 to 4, wherein the storage liquid is a crystalloid liquid. 6.The procedure or process of claim 5 wherein the crystalloid liquid isMedium 199 (Seromed).
 7. The procedure or process of claim 5 wherein thecrystalloid liquid is cardioplegic solution (Bretschneider solution). 8.The procedure or process of claim 5 wherein the crystalloid liquid issupplemented with antibiotics.
 9. The procedure or process of claim 1 to7 wherein the procedure contains a multiple rinsing of the organs and/ortissues before the storage in the same liquid in which the storageoccurs.
 10. The procedure or process of claim 1 to 8 wherein the storageand the outwash of the organs and/or tissues occurres with the sameliquid.
 11. The procedure or process of claim 1 to 9 wherein the storageis done at a pH-value from 3 to
 9. 12. The procedure or process of claim10 wherein the storage is done at a pH-value from 6.9 to 7.8.
 13. Theprocedure or process of claim 10 wherein the storage is done at apH-value from 7.0 to 7.5.
 14. The procedure or process of claim 1 to 12wherein the storage is done at a temperature of 0 to 55° C.
 15. Theprocedure or process of claim 13 wherein the storage is done at atemperature of 0 to 37° C.
 16. The procedure or process of claim 13wherein the storage is done at a temperature of 4° C.
 17. The procedureor process of claim 1 to 15 wherein the storage is done under reducedoxygen pressure.
 18. The procedure or process of claim 16 wherein thestorage is done under anaerobe conditions.
 19. The procedure or processof claim 1 to 15 wherein the storage is done with gases (in the fluid orgaseous stage).
 20. The procedure or process of claim 18 wherein the gasis a rare gas.
 21. The procedure or process of claim 1 to 20 wherein thewashing out occurs pulsating.
 22. The procedure or process of claim 21wherein the washing out of cellular particles, cellular decompositionproducts, as well as soluble substances is done repeatedly.
 23. Theprocedure or process of claim 22 wherein the washing out is done atleast twice, preferably at intervals of 6 weeks.
 24. The procedure orprocess of claim 1 wherein invention-appropriately treated organs andtissues are dried after devitalization and preservation.
 25. Theprocedure or process of claim 1 wherein the organs are hollow organs.26. The procedure or process of claim 1 wherein the washing out isperformed by use of a cultivation apparatus comprising a culture vesselwhich is filled with a liquid (1), two adapters (3) that are connectedwith the two outflows of the culture vessel, a hose, that is connectedwith a pump and a further hose that ends at a stock or dump vessel (5)wherein the pressure gradient Δp is dependent on the riser tube (4) andpressure transducer (8).
 27. The procedure or process of claim 1 whereinthe washing out is done by use of a cultivation apparatus in accordancewith FIG.
 1. 28. The procedure or process of claim 1 wherein the organsand tissues are selected from the group consisting of: Blood vessels,blood vessel valves, lymphatic vessels, lymphatic vessel valves,ureters, bladders, spermatic ducts, bronchial tubes, livers, kidneys,hearts and cardiac valves.
 29. Organs and tissues produced by theprocedure or process of claim 1 to
 27. 30. The procedures or processesfor the production of matrices for the partial or denovo synthesis oforgans and/or tissues comprising the following steps: a. devitalizationand preservation of organs and/or tissues according to the procedures orprocesses of one of the claims 1 to 27, b. cell-repopulation of theorgans and tissues, preferably through re-endothelialization.
 31. Theprocedure or process of claim 29 wherein the organs are hollow organs.32. The procedure or process of caim 30 wherein the hollow organs areselected from the group consisting of: allogenic vessels, to xenogenicvessels and ureter.
 33. Organs produced which are produced according toone of the claims 29 to
 31. 34. The procedure or process of claim 29wherein the cells used for re-population are autologous cells, forexample endothelial cells.
 35. The procedure or process of claim 29wherein the re-endothelialization is done by the seeding of cells on theintraluminal surface of the hollow organs.
 36. The procedure or processof claim 29 wherein a precoating of the hollow organs with adhesionmolecules is performed before cell seeding.
 37. The procedure or processaccording to claim 29, characterized in that the hollow organ isprecoated with autologous serum before seeding of the cells.
 38. Theprocedure or process according to claim 30, characterized in that thehollow organ is precoated with autologous growth and adhesion factorrich serum before seeding of the cells.
 39. The procedure or processaccording to claim 30, further comprising natural and artificial holloworgans selected from the group consisting from: blood vessels, bloodvessel valves, lymphatic vessels, lymphatic vessel valves, ureters,bladders, spermatic ducts, bronchial tubes, hearts and cardiac valves.40. Use of the tissues or organs, available through the procedure orprocess according to one of the claims 1 to 28 in the medicine andveterinarian medicine.
 41. Use of the vessels, available through theprocedure or process according to one of the claims 1 to 28 in thecardiac and vascular surgery.
 42. Use of the vessels, available throughthe procedure or process according to one of the claims 1 to 28 as anaortocoronary bypass or as a vascular transplant in vascularreconstructions.
 43. Use of the vessels, available through the procedureor process according to one of the claims 1 to 28 in peripheral arterialdisease, aneurysms, redooperations in cardiac and vascular surgery andin the case of innate deformations of the vascular system.
 44. Tissuesand organs, available through the procedure or process according to oneof the claims 1 to 28, characterized in that the tissue and organ is inaddition surrounded by a coat of a synthetic material.
 45. Tissues andorgans, available through the procedure or process according to one ofthe claims 1 to 29, characterized in that the tissue and organ is inaddition surrounded by a coat of a synthetic resorbable material. 46.Tissues and organs, available through the procedure or process accordingto one of the claims 1 to 28, characterized in that the tissue and organis in addition surrounded by a coat of a synthetic resorbable material,comprising polyglycon acide.
 47. Cultivation device (bioreactor) for theuse in a procedure or process according to claim 1, comprising a culturevessel filled with a solution (1), two adapters (3) which are connectedwith the two outflows of the culture vessel, a hose, that is connectedwith a computer controlled peristaltic pump and a further hose that endsat an stock or dump vessel (5). The pressure gradient Δp is dependent onthe riser tube (4) and pressure transducer (8).
 48. Culture medium forthe increase of growth, remodeling processes and reduction ofdedifferenziation of vascular cells in tissue culture, characterized inthat autologous growth factors and/or adhesion molecules are added to abasal chemically defined medium or to a full medium.
 49. Culture mediumaccording to claim 48, characterized in that the growth factors and/oradhesion molecules are added in autologous, not heat inactivated serum.50. Culture medium according to claim 48, comprising 5-30% of autologousserum.
 51. Culture medium according to claim 48, comprising 5-20%autologous serum.
 52. Culture medium according to claim 48, comprising10-15% autologous serum.
 53. Culture medium according to any of theclaims 48 to 52, characterized in that additionally recombinant growthfactors are added.
 54. Culture medium according to claim 48,characterized in that autologous growth factors and adhesion moleculesare produced from platelets.
 55. Culture medium according to claim 48,characterized in that autologous growth factors and adhesion moleculesare produced from leucocytes.
 56. Culture medium according to claim 48,characterized in that autologous growth factors and adhesion moleculesare produced from platelets leucocytes.
 57. Culture medium according toclaim 48, characterized in that autologous growth factors and adhesionmolecules are processed from autologous clotted whole blood bycentrifugation technique.
 58. Culture medium according to claim 57,characterized in that the autologous whole blood is stored for at least1 hour at 37° C.
 59. Culture medium according to claim 57, characterizedin that the autologous whole blood is stored for at least for 6 hours at4° C.
 60. Culture medium according to claim 54 and 57, characterized inthat the autologous growth factors and adhesion molecules are gainedfrom enriched (concentrated) platelets.
 61. Culture medium according toclaim 53, characterized in that the recombinant growth factor is bFGF,VEGF, EGF, TGF, Scatter-factor, PDGF or the combination of these growthfactors.
 62. Culture medium according to any of claims 48 to 61, furthercomprising glycosaminoglycane.
 63. Culture medium according to claim 62,characterized in that the glycosaminoglycane is heparine, heparinsulfae,chondroitin, chondroitinsulfate, dermatin or dermatinsulfate. 64.Culture medium according to any of claims 48 to 63, further comprisingtransferrin.
 65. Culture medium according to any of claims 48 to 63,further comprising hydrocortisone.
 66. Culture medium according to anyof claims 48 to 63, further comprising insuline.
 67. Culture mediumaccording to any of claims 48 to 63, further comprising albumine. 68.Culture medium according to any of claims 48 to 63, characterized inthat the culture medium is used for the culture of vascular cells, inparticular endothelial cells, pericytes, pericyte-like-cells and smoothmuscle cells.
 69. Culture medium according to any of claims 47 to 63,characterized in that the culture medium is used for the culture ofnon-vascular cells, in particular from hepatocytes.
 70. Culture mediumaccording to any of claims 48 to 69, characterized in that the culturemedium is used for the precoating of vascular prostheses, cardiac valvesand bypasses in tissue engineering.
 71. Culture medium according to anyof claims 48 to 70, characterized in that the culture medium is usedwithin the framework of tissue engineering.
 72. Culture medium accordingto any of claims 48 to 71, characterized in that the culture medium isused as a preservation solution in tissue banking.
 73. Culture mediumaccording to any one of claims 48 to 72, characterized in that, thatautologous growth factors are gained through mechanical destruction ofbody-own tissues.